Efficacy of novel histone deacetylase inhibitor, AR42, in a mouse model of, human T-lymphotropic virus type 1 adult T cell lymphoma

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) causes a variety of forms of adult T-cell leukemia/lymphoma (ATL), a refractory CD4+/CD25+ T-cell malignancy. Novel approaches to treat ATL patients are required due to the resistance of ATL to conventional chemotherapies. Histone deacetylase inhibitors (HDACi), which induce histone hyperacetylation leading to chromatin remodeling and reactivation of transcriptionally repressed genes have shown efficacy against a variety of cancers. Herein, we tested if valproic acid and the novel orally bioavailable HDACi, AR-42 reduced the proliferation of ATL cell lines by promoting apoptosis and histone hyperacetylation. Both compounds were cytotoxic and elicited a dose dependent increase in cytochrome C and cleaved Poly (ADP-ribose) polymerase (PARP) indicating the induction of cell death by apoptosis and promoted acetylation of histone H3 in both MT-2 and C8166 cell lines. We then evaluated the effects of AR-42, for survival in an ATL NOD/SCID mouse model. A dietary formulation of AR-42 prolonged survival of ATL engrafted mice compared to controls. Our data provide new directions for the treatment of ATL and support the further development of AR-42 against HTLV-1-associated lymphoid malignancies.

Figure 1. Histone deacetylase inhibitors significantly inhibit growth in ATL cell lines

A. MT-2, C8166, and Jurkat T-cells were grown in media only, 1, 2.5, 5 mM valproic acid. At 24, 48, and 72 hours, growth was measured by the addition of MTS reagent and absorbance at 490 nM. B. MT-2, C8166, and Jurkat T-cells were grown in media only, 0.5, 1, 2.5 μM AR-42. At 24, 48, and 72 hours, growth was measured by the addition of MTS reagent and absorbance at 490 nm. C. Immunoblot analysis of HTLV-1 Tax expression in MT-2 and C8166 cells exposed to VPA and AR-42.

Figure 1. Histone deacetylase inhibitors significantly inhibit growth in ATL cell lines

A. MT-2, C8166, and Jurkat T-cells were grown in media only, 1, 2.5, 5 mM valproic acid. At 24, 48, and 72 hours, growth was measured by the addition of MTS reagent and absorbance at 490 nM. B. MT-2, C8166, and Jurkat T-cells were grown in media only, 0.5, 1, 2.5 μM AR-42. At 24, 48, and 72 hours, growth was measured by the addition of MTS reagent and absorbance at 490 nm. C. Immunoblot analysis of HTLV-1 Tax expression in MT-2 and C8166 cells exposed to VPA and AR-42.

Figure 2. Western blot assay indicating induction of cell death by apoptosis and promoted acetylation of histone H3 in ATL cell lines

MT-2 cells were treated with media only, 1, 2.5, 5 mM valproic acid or 0.5, 1, 2.5 μM AR-42 and tested at 24 hours following treatment. Cells showed a dose dependent increase in cleaved PARP, cytochrome C, and acetylated histone H3. C8166 cells were treated with media only, 1, 2.5, 5 mM valproic acid or 0.5, 1, 2.5 μM AR-42. Cells showed a dose dependent increase in cleaved PARP, cytochrome C, and acetylated histone H3.

Figure 3. Diagram of dosing schedule

In phase I, mice were treated by gavage with 50mg/kg AR-42 of vehicle three times per week for two weeks, rested one week and treated in a similar manner for two weeks. In phase II, the mice were started on AR-42 or vehicle in diet at day 21.

Figure 4. Engraftment of MET-1 cells in multiple organs

Examples of NOD/SCID mice inoculated intraperitoneally with 2×10⁷ MET-1 cells. Neoplastic round cells in the brain (meninges), stomach, heart, lung, kidney, and liver. All tissues magnified 200X, bar denotes 50 μm.

Figure 5. Daily feed intake

Mice were fed a diet containing OSU-HDAC42 or control diet ad lib. Average intake per mouse per day did not differ between the groups. In both instances, the mice were allowed to survive until removal criteria were achieved.

Figure 6. Dietary AR-42 enhanced survival in the NOD/SCID ATL mouse model

A.) Serum IL-2Rα levels of mice during the Phase II study. Solid squares indicate values for mice administered vehicle only. Open circles indicate values for mice administered AR-42. B.) Kaplan Meier curve for Phase II in vivo experiments. The solid line represents control mice. Dotted line represents prolonged survival AR-42 treated group.